ES2227771T3 - PURIFIED ALFA-AMYLASE OF FUNGIC ORIGIN STABLE IN ACID. - Google Patents

Abstract

The present invention describes a simple and inexpensive purification method for acid-stable fungal alpha-amylase wherein the alpha-amylase is obtained without significant loss of enzyme activity. The purified acid-stable alpha-amylase which is obtained is substantially free from glucoamylase. The method for obtaining an acid-stable alpha-amylase comprises the steps of adjusting the pH of the enzyme containing solution to a value between 1 and 8, heating the solution to a temperature and for a time sufficient to inactivate glucoamylase, removing the denatured glucoamylase. The purified acid-stable alpha-amylase is used for the conversion of starch. The purified alpha-amylase is also used in an immobilized form.

Description

Purified alpha-amylase of origin Fungal stable in acid.

Technical field

The present invention relates to a Stable fungal alpha-amylase in acid medium purified and to a process to obtain said purified alpha-amylase. In addition, the use is demonstrated of free form purified alpha-amylase and immobilized to obtain specific glucose syrups.

Background of the invention

Stable alpha-amylases in acid medium of fungal origin have long been discovered. For example, A.niger's acid-stable alpha-amylase has been known for more than 30 years (Y. Minoda, K. Yamada, Agr. Biol. Chem., 27 (11), 806-811 (1963) ) and has been thoroughly characterized by Minoda, Yamada and collaborators. The same authors showed the stability of the enzymatic activity in the presence of Ca 2+. The presence of an unstable alpha-amylase in acid medium in preparations of Aspergillus niger by the same authors was described. The acid-stable alpha-amylase enzyme has an optimal pH between 3 and 4 and the optimum temperature is in the range of 70 to 75 ° C.

Several procedures have been developed to obtain stable alpha-amylase in acidic form purified. One method (Minoda and Yamada, cited above) uses fractional precipitation with ammonium sulfate, rivanol and acetone for Get the enzyme in crystalline form. It was found that this crystalline alpha-amylase was contaminated with a unstable alpha-amylase in acidic medium while Glucoamylase and transglucosidase were eliminated. The unstable alpha-amylase in acid medium might subsequently be removed by treating the mixture of alpha-amylase at acidic pH (pH 2.5) and 37 ° C, followed by fractionation. Additional purification was carried out by recrystallization in acetone and gel filtration with Sephadex G-50.

Other publications describe a purification method based on chromatography on DEAE-Sephadex A-25 (DS Chong, Y. Tsujisaka, J. Ferment. Technol. , 54 (4), 264-266 (1976)) or precipitation with ammonium sulfate followed by chromatography on DEAE-Sephadex A-50 (N. Ramasesh, KR Sreekantiah, VS Murthy, Starch, 34 (8), 274-279 (1982)). Sephadex G-25 has also been used, followed by Sepharose Q Fast Flow chromatography (YY. Linko, XY Wu, Biotechnology Techniques, 7 (8)).

European Patent Application EP 0138428 describes the production of an alpha-amylase that is free of transferase and amyloglucosidase. This is achieved by treating the appropriate Aspergillus niger with mutagenic agents and selecting mutant varieties that do not produce unwanted enzymatic activities. The fermentation broth of these mutant varieties then mainly contains the activity of stable alpha-amylase in acidic medium.

The dextrination and saccharification properties of stable alpha-amylase in A. niger acid medium have been fully disclosed. The conversion of liquefied starch into glucose using a mixture of glucoamylase and stable alpha-amylase in acidic medium is described in European Patent Application 0140410 where it is shown that the presence of stable alpha-amylase in acidic medium reduces the time of saccharification and gives higher dextrose yields.

Also the document by Linko et al . (cit. above) describes the use of stable alpha-amylase in acidic medium in the production of dextrose.

Hansen (TT Hansen, New Approaches to Research on Cereal Carbohydrates, eds. RD Hill and L. Munck, Elsevier Science Publishers BV, Amsterdam 1985, 211-216) has disclosed the use of stable alpha-amylase in acid medium for saccharification of a maltodextrin of DE 12 to obtain a syrup with 7% glucose, DP2 48%, DP3 26% and DP4 + 20% after 96 hours. The production of a DE 63 syrup from an acid liquefied syrup of DE 42 by a stable alpha-amylase in immobilized acid medium has been described in the same document. This document however does not describe how stable alpha-amylase was purified in acidic medium that is used. The DE (equivalent of dextrose) is a measure for the number of reducing groups that are present in the molecules. Pure glucose has a DE of 100 and undegraded starch has a DE of 0. Liquefaction of starch with stable alpha-amylase in acidic medium at 75-85 ° C gave starch substrates that were saccharified by glucoamylase. The syrups obtained had the correct yield of dextrose, but were positive starch and had poor filterability.

It has been described in the European Patent Application EP 0157638 the use of stable alpha-amylase in medium immobilized acid obtained from mutant strains.

A suspension of starch, pre-liquefied with B. licheniformis alpha-amylase , with stable alpha-amylase in acid medium at 90 ° C and pH 5, was incubated, given the potential to be used as a post-liquefaction enzyme for production of syrup with high maltose content where a low initial DE is required. After 20 minutes, the temperature was lowered to 60 ° C and barley beta-amylase was added together with pululanase. The final syrup contained 0.5% glucose and 71% maltose and 91% fermentable sugars. A reference syrup produced without alpha-amylase had the same composition of DP1 and DP2, but only 77% fermentable sugars.

A stable alpha-amylase in A.niger acid medium capable of degrading unrefined starch has been described (BN Okolo, LI Ezeogu, CN Mba, J. Sci. Food. Agric. , 69 , 109-115 (1995)). Stable alpha-amylases in acidic medium can not only be found in Aspergillus niger , they have also been found in Aspergillus awamori (RS Bhella, I. Altosaar, Can. J. Microbiol., 31 , 149-153 (1985)) was described that these alpha-amylase were stable between pH 3.5 and 6.5.

Although it is evident from the aforementioned literature that stable alpha-amylase in acidic medium of Aspergillus niger , or of other Aspergillus species that produce stable alpha-amylase in acidic medium, is of potential industrial importance, the preparation of stable alpha-amylase in acidic medium of Aspergillus sp. Non-commercial is free of secondary glucoamylase activity. The presence of glucoamylase in commercial preparations of stable alpha-amylase in acid medium significantly reduces the range of applications where these preparations can be used.

Compendium of the invention

The present invention describes a method of simple and economical purification for alpha-amylase fungal stable in acid medium in which the alpha-amylase is obtained without significant loss of enzymatic activity.

The present invention describes a stable alpha-amylase in purified acid medium that is substantially free of glucoamylase. Preferably, the alpha-amylase is of fungal origin, in which the preferred fungus is an Aspergillus species, preferably Aspergillus niger or Aspergillus awamori .

The present invention also describes a method. to obtain a stable alpha-amylase in acid medium which comprises the stages of

- adjust the pH of the solution containing the enzyme at a value between 1 and 8,

- heat the solution to a temperature specific and for a sufficient time to inactivate the glucoamylase,

- eliminate denatured glucoamylase.

In a preferred embodiment of the invention the heating is carried out in the presence of calcium ions. He heating is at a temperature between 40 to 80 ° C preferably between 50 and 75 ° C.

The present invention further describes the use of a stable alpha-amylase in purified acid medium for the conversion of starch. The purified enzyme of the present invention is used to prepare high maltose syrups or high maltotriose syrups that contain low amounts of glucose, which is less than 10%, preferably less than 5%. The purified alpha-amylase also It is used in immobilized form.

Detailed description of the invention

The present invention describes a heat treatment of the culture broth containing alpha-amylase / glucoamylase obtained after growth of a fungus, preferably of an Aspergillus sp . at specific pH values. The pH and temperature are chosen to reduce or eliminate glucoamylase activity in these preparations. The deactivated glucoamylase flocculates after this treatment and can be easily filtered or removed by centrifugation or other separation technique and thus yield a stable alpha-amylase preparation in pure acid medium. The acid-stable alpha-amylase thus obtained can be used as such or after concentration and addition of stabilizing agents.

Since during this heat treatment alpha-amylase activity is also destroyed unstable in acidic medium, what you get is a solution that contains only stable alpha-amylase in acidic medium and which is free of glucoamylase, alpha-amylase unstable in acidic medium and transglucosidase. The solution can be use as such or can be concentrated before use by filtration, heating or lyophilization. Agents are added stabilizers if necessary.

The present invention describes a stable alpha-amylase in purified acid medium that is substantially free of glucoamylase. Preferably the alpha-amylase is of fungal origin, in which the fungus is an Aspergillus species, preferably Aspergillus niger or Aspergillus awamori .

The present invention also describes a method. to obtain a stable alpha-amylase in acid medium which comprises the stages of

- adjust the pH of the solution containing the enzyme at a value between 1 and 8,

- heat the solution to a temperature specific and for a sufficient time to inactivate the glucoamylase,

- eliminate denatured glucoamylase.

In a preferred embodiment of the invention the solution containing the enzyme is a fermentation broth. First the cellular or microbial material is removed, for example, by filtration or centrifugation. Then the fermentation broth It heats up. The heating is at a temperature where the glucoamylase is inactivated while at the same time the Stable alpha-amylase in acidic medium maintains its activity. The exact temperature depends on the source of the enzyme and of the composition of the fermentation broth. They have been used temperatures between 50 and 80 ° C but preferred temperatures are in the range from 60 to 75 ° C. When the temperature is too much high influences the residual activity of the stable alpha-amylase in acid medium, therefore You must be careful not to overheat the preparation. Preferably the heating is carried out in the presence of calcium ions Calcium ions must be present in a sufficient amount to stabilize the Stable alpha-amylase in acid medium. They have been used satisfactorily amounts of calcium ions in the range of between 50 and 350 ppm. The exact amounts could depend on the source of the enzyme and possible contaminants in the broth of fermentation.

It has been found that values between 150 and 250 ppm were particularly useful.

It is also possible to use the present method with Enzymatic preparations such as those supplied by suppliers because they contain a considerable amount of glucoamylase. In this case it is clear that the method of the present invention does not start with the fermentation broth but with the preparation enzymatic as supplied by the manufacturer from which it has already been removed the microbial material and that is usually buffered and contains stabilizing agents. These preparations too They contain enzymes in a much more concentrated form.

The present invention further describes the use of a stable alpha-amylase in purified acid medium for the conversion of starch. With the purified enzyme it is done possible to prepare high maltose syrups containing relatively low amounts of glucose, that is, the amount of glucose is less than 10% preferably less than 5%.

The purified alpha-amylase is It can be used as such or it can be used in immobilized form. The immobilization can be carried out with techniques of known immobilization. The enzyme can, for example, be trapped in a matrix or gel (alginate, carrageenan) or can be attached to ion exchange resins.

It was found that the products of commercially available alpha-amylase contained a considerable amount of enzymatic contamination. Especially, it was found that the preparations of acid-stable alpha-amylase obtained from fungal origin contained considerable glucoamylase activities and of unstable alpha-amylase in acidic medium.

It was found that the starting enzyme for Examples 1, 2 and 5 (available from Stern Enzyme) contained 7.2 UGA / g and 4,500 UAA / g. An additional preparation sold as G-ZYME? G998 by Enzyme Bio-Systems (used in Examples 3 and 4) contained 15.7 UGA / g and 840 UAA / g. Other enzyme preparations that have been investigated were SANACTASE (powder, Meiji Seika Kaishi Ltd) 1,380 UAA / g and 123 UGA / g and MULTIFRESH (spray-dried variant of G998, Enzyme Bio-Systems) 119 UGA / g and 9,356 UAA / g.

In Example 1 it is shown that incubation at 70 ° C for 1 hour in the presence of calcium ions gives inactivation complete glucoamylase while remaining 74% of the Stable alpha-amylase activity in acidic medium. TO 65 ° C the residual activity of alpha-amylase is approximately 10% higher, also however, glucoamylase It is not completely inactivated as demonstrated in Example 2. Example 3 confirms the result of Example 1 for a second enzymatic preparation When calcium ions are not added to the Preparation of Example 4 the activity of alpha-amylase decreases very strongly.

In Example 5 it is shown that pH influences the speed and amount of inactivation of the alpha-amylase and glucoamylase. In Example 6, determines the amount of unstable alpha-amylase in acidic medium in the G-ZYME enzyme preparation.

Example 7 demonstrates that the Stable alpha-amylase in purified acid medium is use to get high maltose syrups or even high maltotriose syrups containing less than 10% of glucose preferably less than 5%. This is not possible with the Enzyme preparations commercially available.

Example 8 shows that it is possible to obtain syrup of DE 62 starting from a syrup of DE 42 when the Stable alpha-amylase in purified acid medium is use in an immobilized form.

Examples

The determination of enzymatic activities is by standard methods.

Generally as provided by the enzyme supplier.

Glucoamylase activity is determined by para-nitrophenyl-alpha-D-glucopyranoside hydrolysis (KA Holm, Analyst , 3 , 927-929 (1986)). Basically, the procedure is as follows. It is allowed to react to an enzyme preparation under standard conditions (pH 4.2.55 ° C) with para-nitrophenyl-alpha-D-glucopyranoside. In the presence of a glucoamylase this substrate degrades to glucose and para-nitrophenolate which has a yellow color. The amount of para-nitrophenolate produced is determined colorimetrically. Enzymatic activity is calculated from a standard curve that expresses the relationship between para-nitrophenolate concentration and absorbance.

The activity of alpha-amylase is determined by reacting the enzyme with a standard starch solution. The amount of alpha-amylase activity is measured by the rate at which the starch iodine staining capacity decreases. (GB Manning, LLCampbell, J. Biological Chemistry , 236 , 2952-2957 (1961)).

Example 1

1g of powder containing a alpha-amylase / glucoamylase (4,500 UAA / g, 7.2 UGA / g, 200 mg of protein / g of powder), from Stern Enzyme, in 19 g of buffer 0.05 N NaAc / HAc (Ac = acetate) of pH 4.2 containing 200 ppm of Ca 2+. After filtration to obtain a clear liquid, the solution was incubated at 70 ° C for a certain time. At intervals regular heat treatment was stopped by the addiction of 1 ml samples of the solution incubated at 9 ml of a buffer 0.05 N NaAc / HAc at pH 5 and room temperature. The GA and AA activities in these solutions. The results are Show in the table. Typically the protein content of the stable alpha-amylase in remaining acidic medium was Only 20% of the original protein content.

Incubation time (h) % AA activity % activity of GA total* remainder remainder 0.5 77 0.015 one 74 0 1.5 64 0 2.0 fifty 0 3.0 40 0 4.4 25 0  \ begin {minipage} [t] {157mm} * Total AA (alpha-amylase) activity = sum of activities of stable alpha-amylases in acid medium and unstable in acidic medium. \ end {minipage}

It is clearly shown that after a short incubation at pH 4.2 and 70 ° C destroys all activity of glucoamylase

Example 2

1 g of powder containing dissolved alpha-amylase / glucoamylase (4,500 UAA / g, 7.2 UGA / g, 200 mg of protein / g of powder) from Stern Enzyme, in 19 g of buffer 0.05 N NaAc / HAc pH 3.5 containing 200 ppm of Ca 2+. After filtration to obtain a clear liquid the solution is incubated at 65 ° C for a certain time. At regular intervals the heat treatment was stopped by filtration of 1 ml samples of the incubated solution and the subsequent addition to 9 ml of a buffer 0.05 N NaAc / HAc at pH 5 and room temperature. The GA and AA activities in these solutions. The results are Show in the table. Typically the protein content of the stable alpha-amylase in remaining acidic medium was Only 20% of the original protein content.

Incubation time (h) % AA activity % activity of GA total* remainder remainder 0.7 100 16 1.1 88 16 1.6 88 14 2.1 90 8.2 2.5 90 8.6 3.0 82 8.1 3.5 84 8.9  \ begin {minipage} [t] {157mm} * Total AA (alpha-amylase) activity = sum of activities of stable alpha-amylases in acid medium and unstable in acidic medium. \ end {minipage}

It is clearly shown that incubation at pH 3.5 and 65 ° C decreases most glucoamylase activity.

Example 3

1g of G998 (a preparation of stable commercial amylase / glucoamylase in liquid acid medium of Enzyme Bio-Systems, 15.7 UGA / g, 840 UAA / g, 38 mg of proteins / g of enzymatic solution) in 19 g of NaAc / HAc buffer 0.05 PH 3.5 N containing 200 ppm of Ca 2+. After filtration for obtain a clear liquid the solution was incubated at 65 ° C for a certain time. At regular intervals the treatment thermal was stopped by the addition of 1 ml samples of the solution incubated at 9 ml of a 0.05 N NaAc / HAc buffer at pH 5 and temperature ambient. GA and AA activities were determined in these solutions The results are shown in the table. Typically the stable alpha-amylase protein content in remaining acidic medium was only 20-25% of original protein content.

Incubation time (h) % AA activity % activity of GA total* remainder remainder 0.7 98 6.2 one 97 1.4 1.5 80 1.1 2 75 0.2 2.5 76 0.0 3.5 76 0.0  \ begin {minipage} [t] {157mm} * Total AA (alpha-amylase) activity = sum of activities of stable alpha-amylases in acid medium and unstable in acidic medium. \ end {minipage}

Also in this example the Stable alpha-amylase in acid medium is released easily from the activity of contaminating glucoamylase.

Example 4

The experimental conditions used in the Example 4 are the same as those described in Example 3, except that Ca 2+ was not added to the NaAc / HAc buffer at pH 3.5.

Incubation time (min) % AA activity % activity of GA total * remaining without Ca 2+ remnant with Ca 2+ 10 49 twenty 32 30 29 40 twenty-one 98  \ begin {minipage} [t] {157mm} * Total AA (alpha-amylase) activity = sum of activities of stable alpha-amylases in acid medium and unstable in acidic medium. \ end {minipage}

From these figures it is clear that the presence of Ca 2+ is necessary to increase stability stable alpha-amylase thermal medium acid.

Example 5

0.5 g of powder containing dissolved alpha-amylase / glucoamylase (4,500 UAA / g, 7.2 UGA / g, 200 mg of protein / g of powder), from Stern Enzyme, in 2.5 ml of water demineralized After filtration to obtain a liquid transparent, a desalination stage was carried out in a PD-10 column (Pharmacia), yielding 3.5 ml of solution containing alpha-amylase / glucoamylase. This solution was diluted four times with 0.1 N NaAc / HAc buffer. pH 4.2 or 3.5 containing 200 ppm of Ca 2+. The solutions like this Preparations were incubated at 70 ° C for a certain time. After regular intervals the heat treatment was stopped by the addition of 1 ml samples of the solutions incubated to 9 ml of a buffer 0.05 N NaAc / HAc at pH 5 and room temperature. The GA and AA activities in these solutions. The results are Show in the table. Typically the protein content of the stable alpha-amylase in remaining acidic medium was Only 20% of the original protein content.

pH Incubation time (h) % of activity From aa % activity of GA total* remainder remainder 3.5 0.08 84 0 4.2 0.17 100 55 4.2 0.25 80 10  \ begin {minipage} [t] {157mm} * Total AA (alpha-amylase) activity = sum of activities of stable alpha-amylases in acid medium and unstable in acidic medium. \ end {minipage}

Example 6 Determination of the amount of alpha-amylase unstable in acidic medium

To establish the amount of unstable alpha-amylase in acidic medium in an alpha-amylase preparation of Aspergillus sp . commercially, the following method was applied: a solution of the enzyme was brought to pH 2.5 and incubated at 37 ° C for 30 minutes. Then it was cooled and neutralized to pH 4.8. Subsequently, alpha-amylase activity was determined.

Enzyme % from unstable alpha-amylase activity in acidic medium in the enzymatic preparation original G998 5 Enzyme Stern 8

Since the relative AA activities remnants are compared to total AA activity in the examples 1-5, this means that the activity of Stable alpha-amylase in the remaining acidic medium is more high than the values given in the table in the examples 1-5.

Example 7

A spray-dried maltodextrin of DE 5 (equivalent of dextrose) (C \ {76} PUR 1904, made from the complete liquefaction of corn with thermostable α-amylase of Bacillus licheniformis ) was saccharified with G998 or with the \ purified alpha-amylase obtained from G998 as described in example 3.

The saccharifications were performed at 60 ° C and pH 4.5 and at a substrate concentration of 30 g / 100 g of solution.

In the case of saccharification with G998, it added to the saccharification mixture 0.1% on dry matter of G998. For saccharification with α-amylase  purified, the same equivalent of units was added α-amylase as present in 0.1% d.s G988

The following results were obtained:

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6

It is evident from this table that the purified α-amylase produces a spectrum of totally different product than the original G998, which is a mixture of α-amylase activities and glucoamylase Using purified alpha-amylase from The present invention is possible to obtain maltose syrups (DP2) containing low amounts of glucose (DP1). The Amount of glucose is less than 10%.

Example 8

8,900 UAA of G998 were contacted with 10 ml of a wet ion exchanger. The mixture was stirred for 12 h at room temperature and then washed with demineralized water. The conjugate was introduced in a thermostatted glass column equipped with a double jacket and a syrup of DE 42, it was brought to pH 4.5 and was passed through the column at 50 ° C.

Another conjugate was prepared by the addition of 8,900 U of purified α-amylase, as obtained in example 3, 10 ml of the same wet ion exchanger as used for the immobilization of G998. The mixture was stirred. for 12 h at room temperature, and then washed with water demineralized The conjugate was introduced in a glass column thermostat equipped with a double shirt and a syrup of DE 42, it was brought to pH 4.5 and passed through the column at 50 ° C.

The objective was to produce a syrup of DE 62. It They obtained the following results:

7

It is directly evident from the table that the stable α-amylase action model in G998 immobilized purified acid medium is totally different from the action model given by the immobilized G998.

Claims (8)

1. A method to obtain a stable alpha-amylase in purified acid medium of fungal origin, comprising the following stages: - adjust the pH of the solution containing the enzyme at a value between 1 and 8, - heat the solution to a temperature between 40 and 80 ° C to inactivate glucoamylase, - eliminate denatured glucoamylase. 2. A method according to claim 1, wherein The solution is a fermentation broth. 3. A method according to claim 1, wherein Heat is carried out in the presence of calcium ions. 4. A method according to any one of the claims 1 to 3, wherein the solution or broth of Fermentation is heated to a temperature between 50 and 75 ° C. 5. A method according to claim 1, wherein the fungus is a species selected from the genus Aspergillus , preferably Aspergillus niger or Aspergillis awamori . 6. A method according to any one of the claims 1 to 5, wherein the alpha-amylase stable in acidic medium is used for the conversion of starch. 7. A method according to claim 6, wherein stable alpha-amylase in purified acid medium It is used for the preparation of high maltose syrups or high maltotriose syrups that contain less than 10% glucose preferably less than 5% glucose. 8. A method according to any one of the claims 6 and 7, wherein the alpha-amylase stable in purified acid medium is used in immobilized form.